Coupling reaction products of diazotized 2-amino-6-[(2-substitued ethyl)sulfonyl]naphthalene-1-substituted acid with 4-({4-chloro-6-[ethyl(3-{[2-(substituted oxy)ethyl]sulfonyl}phenyl)amino]-heteromonocycl-2-yl}amino)-5-hydroxynaphthalene-2,7-disubstituted acid and their sodium and potassium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-11 till 2010-02-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: "Kanpoan No. 287 -- Environment Protection Agency" "Eisei No. 127 -- Ministry of Health &Welfarew "Heisei 09110131 Kikyoku No. 2 -- Ministry of International Trade & Industry"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- public name of test material : Coupling reaction on diazotized (aminonaphthalen)sulfonyl)ethanol polysulfanate with 6-chloro-N-ethyl-N-(3-(ethylsulfonyl)phenyl)-N'-naphthalen-1-yl-1,3,5-triazine polysulfonate, polyhydroxide, polyamine, sodium and potassium salts
- Substance type: textile dyestuff
- Physical state: red powder
- Analytical purity: ca. 63 % of all colored components
- Lot/batch No.: BOP 07-09
- Expiration date of the lot/batch: 2014-07-31
- Storage condition of test material: At room temperature at about 20 °C

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

deionised water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: without metabolic activation: sodium azide (TA1535, TA100), 4-nitro-o-phenylene-diamine (TA 1537, TA 98), methyl methane sulfonate (WP2 uvrA); with mitabolic activation: 2-aminoanthracene (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approx 72h

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method:reduction in the number of revertants (below the indication factor of 0.5)

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

A biologically relevant increase in revertant colony numbers were observed following treatment with the test material in strains TA 98 and TA 100 (table 1).
The number of colonies in strain TA 98 exceeded the threshold of twice the number of the corresponding solvent control at concentrations 1000 µg/plate and above in the absence of metabolic activation and at 5000 µg/plate in the presence of metabolic activation.
In strain TA 100, the number of colonies exceeded the threshold of twice the number of the corresponding solvent control at concentrations 1000 µg/plate and above in the absence of metabolic activation.
A slight and dose dependent increase in the number of revertants was also detected at 5000 µg/plate in strain TA 1537 in the absence of metabolic activation and in strain TA 100 with metabolic activation but the threshold was not quite reached.

With metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data (table 2) in strain TA 1537 (solvent control). Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of results

Metabolic activation	Test group	Dose level [µg/plate]	Revertant colony counts (mean ± SD)
			TA1535	TA1537	TA98	TA100	WP2uvrA
without	water		12±5	8±2	30±4	122±12	65±12
	untreated		15±2	7±3	25±4	141±3	57±8
	test material	3	12±3	10±5	23±1	122±18	56±4
		10	15±3	11±3	30±4	136±18	64±12
		33	13±1	12±3	28±4	145±24	57±1
		100	11±3	10±3	28±2	151±18	56±1
		333	15±4	8±1	33±5	197±17	62±10
		1000	12±2	9±1	76±4 *	300±7 *	59±8
		2500	14±2 d	11±3 d	112±8 d *	411±30 d *	79±2 d
		5000	11±2 d	19±5 dm	145±10 dm *	408±18 dm *	83±6 dm
	NaN3	10	1919±43 *			2033±44 *
	4-NOPD	10			234±2 *
	4-NOPD	50		145±27 *
	MMS	3.0					1373±35 *
with	water		13±3	8±2	35±7	163±11	72±8
	untreated		21±1	11±2	40±3	153±6	67±8
	test material	3	15±5	13±2	33±3	136±13	52±4
		10	19±4	12±4	41±6	145±12	64±21
		33	15±3	11±2	36±7	164±23	66±7
		100	17±3	9±5	39±4	145±12	75±12
		333	18±3	8±1	32±2	174±22	69±10
		1000	11±6	8±2	36±5	186±6	76±10
		2500	12±2 d	10±3 d	54±3 d	235±17 d	76±7 d
		5000	12±4 d	13±1 dm	88±8 dm *	303±17 dm	81±9 dm
	2-AA	2.5	449±18 *	300±45 *	2223±66 *	2602±105 *
	2-AA	10					381±184 *

NaN3: sodium azide, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene,

d: densely coloured plate

m: manual count

* Value exceeds the respective threshold of twice (TA98, TA100, WP2uvrA) or thrice (TA1535, TA1537) the respective solvent control

Table 2: Laboratory’s historical control data from January 2008 until October 2008 representing approx. 600 experiments (WP2 uvrA the historical data are based on approx. 300 experiments).

Strain	Control	without S9				with S9
		Mean	SD	min	max	Mean	SD	min	max
TA1535	solvent	17	5.17	9	39	21	5.82	8	41
	negative	17	5.33	9	38	20	6.23	10	46
	positive	2024	315.8	1041	3138	294	140.0	102	945
TA1537	solvent	13	3.12	6	25	17	3.90	9	35
	negative	13	3.38	5	26	18	4.05	8	31
	positive	116	30.52	68	407	204	69.54	72	454
TA98	solvent	30	5.59	13	59	39	6.34	20	60
	negative	31	5.45	16	55	39	6.53	19	59
	positive	489	169.8	211	1694	1455	463.0	200	3553
TA100	solvent	130	18.79	89	224	155	22.54	92	218
	negative	139	17.30	93	205	147	21.78	92	234
	positive	2160	342.7	588	3379	1839	621.27	404	3868
WP2uvrA	solvent	49	6.02	33	82	60	8.76	34	89
	negative	49	8.58	30	79	60	8.71	31	84
	positive	986	482.0	187	2367	414	158.56	164	1597

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation
negative with metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and frameshifts in the genome of strains TA 98 and TA 100.
Therefore, the test material is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test material to induce gene mutations in the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA according to OECD Guideline 471 and EC method B13/14.

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

A biologically relevant increase in revertant colony numbers were observed following treatment with the test material in strains TA 98 and TA 100. The number of colonies in strain TA 98 exceeded the threshold of twice the number of the corresponding solvent

control at concentrations 1000 µg/plate and above in the absence of metabolic activation and at 5000 µg/plate in the presence of metabolic activation. The number of colonies in strain TA 100 exceeded the threshold of twice the number of the corresponding solvent control at concentrations 1000 µg/plate and above in the absence of metabolic activation. A slight and dose dependent increase in the number of revertants was also detected at 5000 µg/plate in strain TA 1537 in the absence of metabolic activation and in strain TA 100

with metabolic activation but the threshold was not quite reached.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes and frameshifts in the genome of strains TA 98 and TA 100.

Therefore, the test material is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.